Does leaf wax isotopic characterisation of gymnosperms and angiosperms capture environmental gradients in Himalayas?

The responses of angiosperms and gymnosperms to environmental variables (e.g., rainfall [MAP], temperature [MAT] and pCO₂) remain ambiguous and require systematic investigation. This study monitored the plants' responses to changing environments and measured variations in molecular n-alkane indices, δ¹³C_bulk, δ¹³C_n-alkanes, and δ²H_n-alkanes values in woody gymnosperms (n = 36) and angiosperms (n = 17) from an altitudinal gradient (0.9 to 3.8 km) in the Himalayas The results indicate that δ¹³C_bulk values in angiosperms show a moderate correlation with altitude, MAP, and pCO₂, while gymnosperms show no response in their δ¹³C_bulk. The δ¹³C_n-alkanes values in angiosperms remain unaffected by MAP, MAT, and pCO₂, whereas gymnosperms exhibit a moderate negative correlation. The fractionations between δ¹³C_n-alkanes and δ¹³C_bulk values (ɛ_alk/leaf) are approximately –4.0 ± 1.6 ‰ and –5.6 ± 1.5 ‰ for gymnosperms and angiosperms, respectively. In gymnosperms, the fractionation between δ²H_n-alkane and δ²H_rain (ɛ_alk/rain) is –133.2 ± 39.8 ‰ and –122.8 ± 38.0 ‰ (C₃₁), while in angiosperms, it is –88.5 ± 44.6 ‰ (C₂₉) and –62.4 ± 22.9 ‰ (C₃₁). The δ²H_n-alkane values in gymnosperms and angiosperms are weakly and positively influenced by MAT and MAP, respectively. We found that gymnosperms are enriched in ¹³C (∼1.5–3.0 ‰) and depleted in ²H (∼56–60 ‰) compared to angiosperms; this pattern is consistent throughout the latitudes sampled in this study. The species-specific isotopic response to environmental factors is primarily driven by lower stomatal conductance, smaller leaf-size, and presence of complex tracheids in gymnosperms compared to angiosperms. The statistical K-means algorithm of dual isotope analysis (δ¹³C_n-alkanes and δ²H_n-alkane) provided two distinct clusters with an accuracy of 70 % for angiosperms and gymnosperms, which has implications for studying past vegetation transitions.